Phospho-site receptor mutants reveal distinct roles for phosphorylation in regulating M1 and M4 receptor intracellular trafficking in CHO K1 cells.

Seema Rajani1, Adrian Butcher2, Rachel Thomas1, Andrew Tobin2, Nick Holliday1. 1University of Nottingham, Nottingham, UK, 2University of Leicester, Leicester, UK

Multiple pathways may regulate agonist-promoted endocytosis of Muscarinic receptor subtypes, with differential dependence on G protein receptor kinases, arrestins or clathrin coated pits (Reiner and Nathanson, 2012). In this study we assess the contribution of receptor phosphorylation to the intracellular trafficking of M1 and M4 subtypes in Chinese hamster ovary (CHO K1) cells, using mutants in which all receptor serine / threonine phosphorylation sites have been mutated to alanine (phos-neg).

Stably transfected clonal CHO K1 cell lines expressed N terminal SNAP tagged (ss) murine M1 or M4 receptors, either as wild type (wt) or the phos-neg mutant. Wt and phos-neg clones were matched for cell surface receptor expression using quantified receptor labelling with a membrane impermeant SNAPtag fluorophore (SNAPsurface AF488; Watson et al., 2012). Calcium mobilisation by Fluo4 measurements, and intracellular trafficking studies of SNAPsurface AF488 labelled ssM1 or M4 receptors on a confocal automated IX Ultra platereader, were performed as described (Watson et al., 2012). Agonist-promoted internalisation was assessed by automated granularity analysis of the platereader images, quantifying receptor fluorescence in 6 – 10 µm perinuclear compartments. Concentration response curves were fitted to pooled data with Graphpad Prism v6.0 to yield the indicated pEC50 values.

Acetylcholine (ACh) stimulated calcium mobilisation in both M1 and M4 transfected CHO K1 cells with pEC50 values of 8.22 ± 0.11 (ssM1wt), 8.79 ± 0.12 (ssM1phos-neg), 7.31 ± 0.32 (ssM4wt) and 7.38 ±0.23 (ssM4phos-neg, n = 3 – 4). ACh stimulated rapid ssM1wt receptor clustering followed by internalisation into perinuclear compartments, with a t1/2 of 22 min for 100 µM ACh, and pEC50 of 4.88±0.14 (n=6, measured at 60 min). In contrast ACh did not stimulate internalisation of ssM1phos-neg receptors to these compartments, although some peripheral clustering was observed. Surprisingly, both ssM4wt and ssM4phos-neg receptors underwent rapid internalisation in response to 100 µM ACh (respective t1/2s of 18 and 13 min); indeed ACh was 7-fold more potent in stimulating ssM4phos-neg trafficking to similar intracellular compartments (pEC50 5.76±0.23 at 60min) than for ssM4wt (pEC50 4.92±0.18, each n=3). We examined whether the apparent increase in ACh potency was due to reduced ssM4phos-neg recycling following initial endocytosis. Cells were treated with 100 µM ACh for 1 h, then washed extensively to remove agonist, before monitoring receptor recycling from perinuclear compartments. While substantial recycling of ssM4wt receptors was observed 60 min post wash (24.7 ± 15.4 % of the internalisation at t=0), ssM4phos-neg receptors remained internalised at this time (91.8 ± 5.5 %, P < 0.05 Student’s t-test).

We conclude that there are potentially divergent roles for receptor phosphorylation in controlling M1 and M4 receptor intracellular trafficking. Receptor phospho-sites are required for efficient M1 receptor endocytosis, but are not necessary for M4 receptor internalisation in CHO K1 cells. Instead, global mutation of Ser / Thr phospho sites inhibits M4 receptor recycling, suggesting that previously identified recycling motifs within intracellular loop 3 of this receptor subtype might be controlled by phosphorylation status (Hashimoto et al., 2008).

RLT was supported by an Anne McLaren fellowship from the University of Nottingham.

Hasimoto Y et al, J Pharmacol Exp Ther 325:947, 2008

Reiner C & Nathanson NM, Handbook Exp Pharmacol, 208:61, 2012

Watson SJ et al, Mol Pharmacol 81:631, 2012